Cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19), also designated cyclodextrin glucanotransferase or cyclodextrin glycosyltransferase, in the following termed CGTase, catalyses the conversion of starch and similar substrates into cyclomaltodextrins via an intramolecular transglycosylation reaction, thereby forming cyclomaltodextrins, in the following termed cyclodextrins (or CD), of various sizes. Commercially most important are cyclodextrins of 6, 7 and 8 glucose units, which are termed .alpha.-, .beta.- and .gamma.-cyclodextrins, respectively. Commercially less important are cyclodextrins of 9, 10, and 11 glucose units, which are termed .delta.-, .epsilon.-, and .zeta.-cyclodextrins, respectively. Cyclodextrins are thus cyclic glucose oligomers with a hydrophobic internal cavity. They are able to form inclusion complexes with many small hydrophobic molecules in aqueous solutions, resulting in changes in physical properties, e.g. increased solubility and stability and decreased chemical reactivity and volatility. Cyclodextrins find applications particularly in the food, cosmetic, chemical and pharmaceutical industries.
Most CGTases have both starch-degrading activity and transglycosylation activity. Although some CGTases produce mainly .alpha.-cyclodextrins and some CGTases produce mainly .beta.-cyclodextrins, CGTases usually form a mixture of .alpha.-, .beta.- and .gamma.-cyclodextrins. Selective precipitation steps with organic solvents may be used for the isolation of separate .alpha.-, .beta.- and .gamma.-cyclodextrins. To avoid expensive and environmentally harmful procedures, the availability of CGTases capable of producing an increased ratio of one particular type of cyclodextrin, in particular with respect to .alpha.-, .beta.- or .gamma.-cyclodextrin, is desirable.
WO 96/33267 (Novo Nordisk) describes CGTase variants showing a modified substrate binding and/or product selectivity. Although CGTase variants produced by mutation at positions 47, 145, 146, 147, 196 or 371 have been described, the specific CGTase variants of this invention have never been described or even suggested.